To Margaret C.M. Smith for kindly providing the integrase clone 
 Funding support from: 
 Genome Canada/Genome Prairie 
 NSERC Discovery and Strategic Project grants

1. Production of Culture
<ol>
 <li>Prepare sterile liquid media: TY10 (5 g/l tryptone, 3 g/l yeast extract, 0.44 g/l calcium chloride dehydrate), and LB11 (10 g/l tryptone, 5 g/l yeast extract, 5 g/ sodium chloride, pH 7).</li>
 <li>Inoculate from a single colony: SmUW227 (S. meliloti LP-strain: construction will be described elsewhere, strain construction details available upon request) () into 5 ml of TY media with 50 &mu;g/ml spectinomycin. Inoculate the following strains into 5 ml of LB liquid media, supplementing with the given antibiotic: E. coli DH5&alpha;12 containing pJC2, the integrase expression plasmid, with 10 &mu;g/ml tetracycline; E. coli MT61613, the mobilizer, with 10 &mu;g/ml chloramphenicol; and E. coli DH5&alpha; containing pJH110, the donor cassette plasmid, with 5 &mu;g/ml gentamicin.</li>
 <li>Incubate E. coli strains overnight at 37 &deg;C with constant shaking. Incubate S. meliloti strain at 30 &deg;C for two days with shaking. It is possible to obtain a S. meliloti culture overnight with a larger inoculum (1:500 subculture of a saturated culture). </li>
</ol>
2. Culture Preparation and Mixing
<ol>
 <li>Wash 1.5 ml of culture for each strain by collecting the cell pellet by centrifugation at 17,000 x g for 30 seconds and resuspending in 1 ml sterile 0.85% NaCl; repeat once.</li>
 <li>Resuspend pellet in 100 &mu;l of sterile 0.85% NaCl.</li>
 <li>Individually spot 10 &mu;l of washed culture for each strain on a plain TY agar plate. These spots are control spots.</li>
 <li>Add 40 &mu;l of each strain excluding the E. coli DH5&alpha;12 containing pJC2 in a sterile tube, mix, and spot 120 &mu;l of this mixture on a plain TY agar plate. This spot is the no-integrase negative control.</li>
 <li>Add 40 &mu;l of each strain in a sterile tube, mix, and spot 160 &mu;l of this mixture on a plain TY agar plate. This spot is the IMCE mating spot.</li>
 <li>Allow spots to dry in a laminar flow hood.</li>
 <li>Seal plates and incubate at 30 &deg;C overnight.</li>
</ol>
3. Isolation of Trans-integrants
<ol>
 <li>
 Streak control spots and IMCE spot onto TY agar plates supplemented with 200 &mu;g/ml streptomycin (SmUW227 is based on Rm1021 which carries a mutation conferring high-level resistance to streptomycin14) and 30 &mu;g/ml gentamicin.</li>
 <li>For calculation of the IMCE efficiency, resuspend approximately &frac14; of the IMCE mating spot in 500 &mu;l sterile 0.85% NaCl. Make 10-fold serial dilutions to 10-7. Plate dilutions 10-2 through 10-5 on TY agar plates supplemented with 200 &mu;g/ml streptomycin and 30 &mu;g/ml gentamicin, to select for trans-integrants. Plate dilutions 10-3 through 10-7 on TY agar plates supplemented with 200 &mu;g/ml streptomycin, to select for both trans-integrants and potential recipients, i.e. total recipients.</li>
 <li>For isolation of markerless trans-integrants resuspend approximately a quarter of the IMCE mating spot in 500 &mu;l of sterile 0.85% NaCl. Make 10-fold serial dilutions to 10-6. Plate dilutions 10-4 to 10-6 on four TY agar plates supplemented with 200 &mu;g/ml streptomycin and 200 &mu;g/ml X-gluc.</li>
 <li>Incubate plates at 30 &deg;C for 3 days.</li>
 <li>View RFP trans-integrants under green light (525 nm) and through a red filter (&gt;610 nm) 15.</li>
 <li>Calculate the percent IMCE efficiency as CFU of trans-integrants compared to the CFU of total recipients. Approximately half of trans-integrants will have undergone true cassette exchange making them white and spectinomycin sensitive.</li>
 <li>To find the markerless trans-integrants (where the donor vector does not contain Gmr like in pJH110) screen for a white colony (an extended incubation period, 1-2 extra days at room temperature, will enhance the differentiation of colonies via X-gluc, this is necessary in S. meliloti SmUW227), confirm the colony's spectinomycin sensitivity by screening on an TY agar plate without antibiotics and an TY agar plate containing 100 &mu;g/ml spectinomycin.</li>
</ol>
4. Representative Results
After three days of incubation on TY supplemented with streptomycin and gentamicin the control streaks should have no growth. The IMCE streak should have confluent growth on the head streak and many colonies on the second streak, as seen in Figure 2. The efficiency of trans-integration, expressed as the percentage of trans-integrants to total recipients, should be in the range of 0.5%. Approximately half of trans-integrants will be spectinomycin sensitive and white showing they have undergone true cassette exchange. Trans-integrants containing the tester rfp donor cassette from pJH110 should display discernable RFP fluorescence when viewed under green light (525 nm) and through a red filter (&gt;610 nm) 15. 
<img src="/files/ftp_upload/3698/3698fig1.jpg" alt="Figure 1"/> 
Figure 1. Conjugation mixture illustration: Aided by the expression of the transfer genes from pRK600, all of the plasmids are transferred randomly from cell to cell. This transfer results in the creation of trans-integrants in the mixture, through the LP-strain's acquisition of the two plasmids required for IMCE, the integrase (int) helper plasmid pJC2 and the donor plasmid pJH110. The donor cassette from the non-replicating donor plasmid (pJH110) is exchanged via &Phi;C31 integrase activity with the markers of the LP-cassette on the chromosome, resulting in the loss of the LP-markers (Spr and uidA) and the maintenance of the donor cassette (rfp and Gmr) in the resulting trans-integrant.
<img src="/files/ftp_upload/3698/3698fig2.jpg" alt="Figure 2"/> 
Figure 2. A: Mating spot plate on a non-selective TY plate showing dried cell mixtures on agar surface. B: Mating spots streaked on streptomycin-gentamicin-X-gluc plate, from top left clockwise no integrase control, IMCE into S. meliloti, E. coli DH5&alpha; containing pJC2 control, E. coli DH5a containing pJH110 control, E. coli MT616 control, and S. meliloti UW227 control. C: 10-2 dilution of mating spot resuspension on TY streptomycin-X-gluc agar. D: 10-2 dilution of mating spot resuspension on TY streptomycin-gentamicin-X-gluc agar (blue colonies are single recombinants, white colonies have undergone true cassette exchange). E: 10-2 dilution of mating spot resuspension on TY streptomycin-X-gluc agar showing lack of fluorescence. F: 10-2 dilution of mating spot resuspension on TY streptomycin-gentamicin-X-gluc agar showing two levels of fluorescence (brighter colonies correspond to blue colonies and have higher rfp expression presumably due to promoter read-though from vector sequence, where colonies having undergone true-cassette exchange contain RFP with only its immediate promoter with no read-through from the lac promoter in the vector, which is absent.).

<ol>
	<li>Itaya, M., Tsuge, K., Koizumi, M., Fujita, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+two+genomes+in+one+cell:+Stable+cloning+of+the+Synechocystis+PCC6803+genome+in+the+Bacillus+subtilis+168+genome.">Combining two genomes in one cell: Stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 15971-15976 (2005).</li><li>Kushtoss, S., Rao, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+Integration+Function+of+the+Streptomycete+Bacteriophage+FC31.">Analysis of the Integration Function of the Streptomycete Bacteriophage FC31.</a> J. Mol. Biol. 222, 897-908 (1991).</li><li>Brown, W. R., Lee, N. C., Xu, Z., Smith, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serine+recombinases+as+tools+for+genome+engineering.">Serine recombinases as tools for genome engineering.</a> Methods. 53, 372-379 (2011).</li><li>Groth, A. C., Olivares, E. C., Thyagarajan, B., Calos, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phage+integrase+directs+efficient+site-specific+integration+in+human+cells.">A phage integrase directs efficient site-specific integration in human cells.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 5995-6000 (2000).</li><li>Rowley, P. A., Smith, M. C., Younger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+motif+in+the+C-terminal+domain+of+FC31+integrase+controls+the+directionality+of+recombination.">A motif in the C-terminal domain of FC31 integrase controls the directionality of recombination.</a> Nuc. Acid. Res. 36, 3879-3891 (2008).</li><li>Campbell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+monomeric+red+fluorescent+protein.">A monomeric red fluorescent protein.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 7877-7882 (2002).</li><li>Schafer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+mobilizable+multi-purpose+cloning+vectors+derived+from+the+Escherichia+coli+plasmids+pK18+and+pK19:+selection+of+defined+deletions+in+the+chromosome+of+Corynebacterium+glutumicum.">Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutumicum.</a> Gene. 145, 69-73 (1994).</li><li>Thorpe, H. M., Smith, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+site-specific+integration+of+bacteriophage+DNA+catalyzed+by+a+recombinase+of+the+resolvase&#47;invertase+family.">In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase&#47;invertase family.</a> Proc. Natl. Acad. Sci. 95, 5505-5510 (1998).</li><li>Jones, J. D., Gutterson, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+mobilizable+cosmid+vector,+pRK7813,+and+its+use+in+a+rapid+method+for+marker+exchange+in+Pseudomonas+fluorescens+strain+HV37a.">An efficient mobilizable cosmid vector, pRK7813, and its use in a rapid method for marker exchange in Pseudomonas fluorescens strain HV37a.</a> Gene. 61, 299-306 (1987).</li><li>Beringer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R+Factor+transfer+in+Rhizobium+leguminosarum.">R Factor transfer in Rhizobium leguminosarum.</a> J. Gen. Microbiol. 84, 188-198 (1974).</li><li>Lennox, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+of+linked+genetic+characters+of+the+host+by+bacteriophage+P1.">Transduction of linked genetic characters of the host by bacteriophage P1.</a> Virology. 1, 190-206 (1955).</li><li>Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+transformation+of+Escherichia+coli+with+plasmids.">Studies on transformation of Escherichia coli with plasmids.</a> J. Mol. Biol. 166, 557-580 (1983).</li><li>Charles, T. C., Finan, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+map+of+Rhizobium+meliloti+megaplasmid+pRmeSU47b.">Genetic map of Rhizobium meliloti megaplasmid pRmeSU47b.</a> J. Bacteriol. 172, 2469-2476 (1990).</li><li>Leigh, J. A., Signer, E. R., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exopolysaccharide-deficient+mutants+of+Rhizobium+meliloti+that+form+ineffective+nodules.">Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective nodules.</a> Proc. Natl. Acad. Sci. U.S.A. 82, 6231-6235 (1985).</li><li>Heil, J. R., Nordeste, R. F., Charles, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluorescence+theatre:+a+cost-effective+device+using+theatre+gels+for+fluorescent+protein+and+dye+screening.">The fluorescence theatre: a cost-effective device using theatre gels for fluorescent protein and dye screening.</a> Can. J. Microbiol. 57, 339-342 (2011).</li><li>Datsenko, K. A., Wanner, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+inactivation+of+chromosomal+genes+in+Escherichia+coli+K-12+using+PCR+products.">One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 6640-6645 (2000).</li><li>Lesic, B., Rahme, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+lambda+Red+recombinase+system+to+rapidly+generate+mutants+in+Pseudomonas+aeruginosa.">Use of the lambda Red recombinase system to rapidly generate mutants in Pseudomonas aeruginosa.</a> BMC Mol. Biol.. 9, 20-20 (2008).</li><li>Choi, K. -. H., Schweizer, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mini-Tn7+insertion+in+bacteria+with+single+attTn7+sites:+example+Pseudomonas+aeruginosa.">mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa.</a> Nat. Protocols. 1, 153-161 (2006).</li><li>Thomason, L. C., Calendar, R., Ow, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+insertion+and+replacement+in+Schizosaccharomyces+pombe+mediated+by+the+Streptomyces+bacteriophage+FC31+site-specific+recombination+system.">Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage FC31 site-specific recombination system.</a> Molecular Genetics and Genomics. 265, 1031-1038 (2001).</li><li>Katzen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gateway+recombinational+cloning:+a+biological+operating+system.">Gateway recombinational cloning: a biological operating system.</a> Expert Opin. Drug Discovery. , 571-586 (2007).</li><li>Charles, T. C., Doty, S. L., Nester, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+Agrobacterium+strains+by+electroporation+of+genomic+DNA+and+its+utility+in+analysis+of+chromosomal+virulence+mutations.">Construction of Agrobacterium strains by electroporation of genomic DNA and its utility in analysis of chromosomal virulence mutations.</a> Appl. Environ. Microbiol. 60, 4192-4194 (1994).</li></ol>

No conflicts of interest declared.

The IMCE technique allows for the efficient integration of a single attB flanked DNA cassette into the LP-locus of a previously engineered strain. Once the desired construct is cloned in place of rfp to create the donor cassette, the technique does not require subsequent DNA purification and transformation, making it very robust. It is critical that appropriate growth controls are included, to be certain the antibiotic resistance is due to the creation of trans-integrants and not other factors.
<p c...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Bioshop Canada Inc.</td>
			<td>STP101</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spectinomycin</td>
			<td>Bioshop Canada Inc.</td>
			<td><a href="https://ssl34.alentus.com/bioshopcanada/detail.asp?Pin=SPE201">SPE201</a></td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Bioshop Canada Inc.</td>
			<td>GTA202</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Choramphenicol</td>
			<td>Bioshop Canada Inc.</td>
			<td>CLR201</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>Bioshop Canada Inc.</td>
			<td>TET701</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Bioshop Canada Inc.</td>
			<td>KAN201</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bacteriological grade agar</td>
			<td>Bioshop Canada Inc.</td>
			<td>AGR001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Bioshop Canada Inc.</td>
			<td>TRP402</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Bioshop Canada Inc.</td>
			<td>YEX401</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Bioshop Canada Inc.</td>
			<td>SOD001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Bioshop Canada Inc.</td>
			<td>CCL444</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>X-gluc</td>
			<td>Gold Biotechnology Inc.</td>
			<td>G1281C1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>E. coli MT616 strain</td>
			<td>Available upon request</td>
			<td>&nbsp;</td>
			<td>Also used outside of our lab</td>
		</tr>
		<tr>
			<td>E. coli pJC2 strain</td>
			<td>In house, available by request</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>E. coli pJH110 strain</td>
			<td>In house, available by request</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SmUW227 strain</td>
			<td>In house, available by request</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

site-specific bacterial chromosome engineering: &#934;c31 integrase mediated cassette exchange (imce)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

Watch this Scientific Journal Video about Site-specific Bacterial Chromosome Engineering: ÃŽÂ¦C31 Integrase Mediated Cassette Exchange IMCE at JoVE.com

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

site-specific-bacterial-chromosome-engineering-c31-integrase-mediated

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15.5K Views.  University of Waterloo. A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the ΦC31 integrase.

Video: Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE)

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea (ureolysis) in naturally occurring environments through secretion of an enzyme urease. This process of ureolysis, through a chain of chemical reactions, leads to the formation of calcium carbonate precipitates. This is known as Microbiologically Induced Calcite Precipitation (MICP). The proper culture protocols for MICP are detailed here. Finally, visualization experiments under different modes of microscopy were performed to understand various aspects of the precipitation process. Techniques like optical microscopy, Scanning Electron Microscopy (SEM) and X-Ray Photo-electron Spectroscopy (XPS)&#160;were employed to chemically characterize the end-product. Further, the ability of these precipitates to clog pores inside a natural porous medium was demonstrated through a qualitative experiment where sponge bars were used to mimic a pore-network with a range of length scales. A sponge bar dipped in the culture medium containing the bacterial cells hardens due to the clogging of its pores resulting from the continuous process of chemical precipitation. This hardened sponge bar exhibits superior strength when compared to a control sponge bar which becomes compressed and squeezed under the action of an applied external load, while the hardened bar is able to support the same weight with little deformation.

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.